Sphingolipids comprise a large class of biologically significant compounds, such compounds being constituents of cell membranes. The principal component of sphingolipids is almost invariably a particular long chain base, sphingosine.
When present in the cell, such sphingolipids are metabolized, yielding sphingosine as a metabolic intermediate. Interest in these metabolic intermediates in general, and sphingosine in particular, was heightened when it was realized that sphingosine was a potent and specific inhibitor of protein kinase C. Hakomori, S., Am. Rev. Biochem. 50, 733-739 (1981). This discovery was significant due to kinase C's known function as a pivotal regulatory enzyme in cell growth. E.g., Merrill, A. H., J. Bioenerg. Biomembr., 23, 83-104 (1991); Hannun, Y. A. et al., Science, 243, 500-506 (1989).
Recent research in the area of sphingosine and its analogs has demonstrated that one such analog has a significant effect on cell growth. More specifically, that analog, S-1-P, has proven to be a very potent mitogen in Swiss 3T3 fibroblasts. Zhang, H. et al., J. Cell Biol. 114,155-167 (1991). S-1-P has further been shown to cause rapid translocation of calcium from intracellular stores, i.e., from IP.sub.3 -sensitive and IP.sub.3 -insensitive intercellular pools in permeabilized smooth muscle cells. Ghosh, T. K., Science, 248, 1653-1656 (1990).
That S-1-P possessed the foregoing properties, i.e., as a mitogen and calcium translocator, was surprising because it was previously thought that the major role of S-1-P was its involvement in the catabolic pathway for sphingosine. Specifically, in that pathway, S-1-P is produced from sphingosine by the action of a specific kinase which is resident in the cell cytoplasm. After its production, S-1-P is cleaved into 2-hexadecenal and ethanolamine-1-phosphate by sphingosine-1-phosphate aldolase, which is resident in the endoplasmic reticulum of the cell. Stoffel, W. et al., Hoppe-Seyer's Z. Physiol. Chem., 349, 635-642 (1970); Van Valdhoven, P. P. et al., J. Biol. Chem., 266, 12502-12507 (1991).
In order to advance the study of S-1-P, and in particular its mitogenic properties and role as a "secondary" messenger in causing calcium translocation, it would be beneficial to be able to synthesize S-1-P on a commercial scale. Known synthesis routes have proven inadequate for this purpose, however, hampering this investigation. In particular, presently available routes are exceedingly time consuming and, further, are only able to provide S-1-P in relatively low yields and/or quantities.
One known method for the preparation of S-1-P is by means of an enzymatic synthesis. This method comprises hydrolyzing sphingosylphosphorylcholine with phospholipase D isolated from Streptomyces chromofuscus. However, this method gives a mixture of D-erythro and L-threo isomers of S-1-P, which are not separable. Moreover, the quantity of S-1-P provided by this method is relatively low, i.e., milligram quantities. Van Veldohoven, P. et al., J. Lipids Res. 30, 611 (1989). In contrast, a recently reported chemical synthesis provides for the preparation of only the D-erythro isomer of S-1-P. This synthesis begins by preparing its "starting" material (a D-erythro-olefinic alcohol) from L-serine. The starting material has both primary and secondary hydroxyl functionalities, as well as an amino functionality. Once the starting material is prepared, the synthesis proceeds by protecting both the amino and secondary hydroxyl functionalities of that material. The primary hydroxyl functionality, which is left unprotected, is then modified by phosphorylation, with the amino and secondary hydroxyl protective groups being subsequently removed to provide the aforesaid S-1-P isomer. While this synthesis provides only a single isomer of S-1-P, the synthesis remains lengthy (10 steps from L-serine to S-1-P, requiring about 98 hours to complete) and provides S-1-P in a relatively low yield (about 10.7%). Ruan, F., Bioorg. & Med. Chem. Let., 2 (9), 973-978 (1992).
A more recent chemical synthesis prepares S-1-P using a particular "starting" compound, namely 3-O-tertbutyldimethylsilyl ("TBDMS") protected D-erythroazidosphingosine ("the azido compound"). More specifically, the azido compound, which has primary and secondary hydroxyl functionalities as well as an azido functionality, is prepared from D-glucose in a nine step process at a yield of 15%, resulting in protection of the secondary hydroxyl functionality by the TBDMS group.
Once the azido compound has been prepared, its amino functionality is also protected using a carbonate compound. The primary hydroxyl functionality of the resulting compound, which remains unprotected, is then phosphorylated using bis(2-cyanoethoxy)(diisopropylamino)phosphine to provide a phosphite intermediate. After oxidation of the intermediate, the groups protecting the secondary hydroxyl and amino functionalities are removed, providing S-1-P. Kratzer, B. et al, Tetra. Let., 34. (11), 1761-1764 (1993).
While this method provides S-1-P, the synthesis is quite lengthy (14 steps from D-glucose to S-1-P, requiring about 318 hours to complete) and provides S-1-P in only a relatively low yield (about 8.4% overall). In addition, the method requires the use of some hazardous chemicals, e.g. dioxane, as well as the use of sodium azide, a compound which is highly explosive, especially in the presence of halogenated solvents.
Returning to the subject of the S-1-P aldolase (which functions to cleave S-1-P into two compounds), the study of its role at the cellular level would be enhanced by the availability of compounds which would inhibit the activity of the aldolase. Such would aid in the isolation and purification of S-1-P aldolase, thus allowing experimentation with that enzyme to be readily undertaken. To date, there is believed to be only a single inhibitor of S-1-P aldolase reported in the literature, that being a sphingosine derivative referred to as dihydrosphingosine-1-phosphonate ("DS-1-P").
The article that discloses DS-1-P attributes its S-1-P aldolase-inhibiting property to the substitution of a --CH.sub.2 --PO.sub.3 group for the ester linkage (--CH.sub.2 --O--PO.sub.3) found on the S-1-P compound. This substitution is said to reduce the distance between the head phosphate group and the remainder of the compound, particularly in regard to the 2C and 3C carbon atoms. The shortening of the compound is said to be significant because it is well known that the steric requirements of S-1-P aldolase are highly specific toward the 2S, 3R configuration of S-1-P. Thus, assuming the function of the phosphate functionality is to bind the compound to a positively-charged group at an enzyme's surface, thereby supporting the correct orientation of the two optically active centers (C2 and C3), the aforedescribed reduction in distance between the phosphate functionality and the active centers should lead to strongly altered binding by electrostatic forces. The end result of this is said to be a reduction in the velocity of the phosphonate and a competitive inhibition of the S-1-P aldolase reaction. Despite DS-1-P's activity as a S-1-P aldolase inhibitor, however, it is unfortunately highly toxic, rendering it useless for experimentation at the cellular level. Stoffel, W. et al., Chem. & Phys. of Lipids, 13,372-388 (1974).
Thus, there exists a need for a process for preparing S-1-P and analogs thereof which is more amenable to production of S-1-P and its analogs on a commercial scale, specifically, by providing those compounds at greater yields and quantities in a shorter period of time, while minimizing the use of hazardous chemicals. Further, a need exists for a compound or compounds which would inhibit the activity of S-1-P aldolase, thereby aiding its isolation and purification as well as contributing to a greater understanding of the functions of that aldolase at the cellular level.
The present invention fulfills the foregoing needs by providing a process for preparing S-1-P and analogs thereof which is more amenable to production of those compounds on a commercial scale. Specifically, the process provides S-1-P (as well as its analogs) at a greater yield and quantity, as well as at a lower preparation time, as compared to known processes, while minimizing the use of hazardous chemicals. The invention further provides a series of non-toxic compounds which function to inhibit the activity of S-1-P aldolase in respect to its cleavage of S-1-P.
These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.